CN112094350B - Monoclonal antibody of mouse anti-cell surface glycoprotein CD326 applicable to tumor cell capture - Google Patents

Monoclonal antibody of mouse anti-cell surface glycoprotein CD326 applicable to tumor cell capture Download PDF

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Publication number
CN112094350B
CN112094350B CN202010484564.2A CN202010484564A CN112094350B CN 112094350 B CN112094350 B CN 112094350B CN 202010484564 A CN202010484564 A CN 202010484564A CN 112094350 B CN112094350 B CN 112094350B
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China
Prior art keywords
antibody
monoclonal antibody
cell
mouse anti
surface glycoprotein
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CN112094350A (en
Inventor
孟逊
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Puzhong Discovery Pharmaceutical Technology Shanghai Co ltd
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Puzhong Discovery Pharmaceutical Technology Shanghai Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2821—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against ICAM molecules, e.g. CD50, CD54, CD102
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The invention relates to a monoclonal antibody, in particular to a monoclonal antibody of a mouse anti-cell surface glycoprotein CD326 which can be applied to tumor cell capture, comprising the following steps: s1, protein QC; s2, animal immunization; s3, serum detection and screening; s4, fusion and screening; s5, subcloning and screening; s6, preparing an antibody supernatant; s7, antibody verification. The monoclonal antibody of the mouse anti-cell surface glycoprotein CD326, which can be applied to tumor cell capture, has the characteristics of high affinity, high specificity, multiple application scenarios and the like, and can be applied to living cell enrichment.

Description

Monoclonal antibody of mouse anti-cell surface glycoprotein CD326 applicable to tumor cell capture
Technical Field
The invention relates to a monoclonal antibody, in particular to a monoclonal antibody of a mouse anti-cell surface glycoprotein CD326, which can be applied to tumor cell capture.
Background
CD326 is also called EpCAM, which has a relative molecular mass of 40ku, expresses glycoprotein on the surface of normal epithelial cells and most malignant epithelial tumor cells of human parts, has various biological functions of regulating cell proliferation, differentiation, migration and the like, is similar to immunoglobulin superfamily adhesion molecules, and can be classified into adhesion molecule families according to the function classification thereof. EpCAM is a type I transmembrane molecule that structurally contains an extracellular domain, a transmembrane region, and an intracellular region. The tumor cells overexpressed by the EpCAM have high proliferation activity and strong invasiveness, and the proliferation or perhaps invasiveness of the tumor cells can be obviously inhibited by downregulating the EpCAM gene through an RNA interference technology. EpCAM has the function of promoting immune escape of Th2 differentiated tumors, and can block dendritic cell major histocompatibility complex ii from limiting antigen presentation, and generate T cells with functional defects, so that T cell-dependent immune response can be escaped, and tumorigenesis can be promoted. There are few antibody products on the market that use CD326 as a target carrier, and no data indicate that it can be used for live tumor cell enrichment. While there is a lack of effective antibody tools for this research area.
Disclosure of Invention
The invention aims to provide a tool for researching targeted proteins, and the deeper research on protein expression changes and protein network patterns of different cancer patients and different stages of cancers is realized.
The technical scheme adopted by the invention is as follows: a monoclonal antibody of a mouse anti-cell surface glycoprotein CD326 applicable to tumor cell capture, the process comprising the steps of:
s1, protein QC: purchasing commercial proteins;
s2, animal immunization: selecting 6 Balb/c mice for antigen immunization, monitoring serum titers of the Balb/c mice to determine optimal immunized mice, performing boosting for 3 to 4 times after primary immunization, detecting titer by taking serum of the mice after boosting, screening out qualified mice with one impact for fusion, and continuing boosting for one to two times until the titer is highest for fusion;
s3, serum detection and screening: immunized mice are subjected to orbital blood collection, and the serum titer is detected by ELISA, so that the qualified serum titer is determined;
s4, fusion and screening: fusing the whole spleen and 1/2 lymph nodes with a myeloma SP2/0 cell line, paving fused cells on 4 384-well plates for culture, collecting supernatants of all the wells, screening immune proteins by ELISA, performing microscopic examination on positive Kong Zhuaidao-well plates with cells for continuous culture, collecting supernatants of all the wells after a few days of growth, detecting reaction with soluble fragment detection antigen by ELISA, further detecting protein binding of different dilutions of positive wells, and performing affinity sequencing on 20 parent clones with highest affinity for each fusion immunogen into subclones;
s5, subcloning and screening: subcloning by limiting dilution method and ELISA screening to obtain monoclonal hybridoma cells, spreading 96-well plates on the cells, culturing to cover the bottom of about 1/6, ELISA detecting the reaction of each well supernatant on immune antigen, taking two wells with high OD value and good cell state to enter the next round of subcloning, repeating the steps until the positive rate of cell strains in the wells is 100%, obtaining monoclonal cell strains at this time, immediately expanding and culturing all positive cells after the last round of subcloning, freezing one part for later use, and preparing the other part of supernatant or ascites;
s6, preparing antibody supernatant: injecting the finally obtained monoclonal cell strain into an F1 mouse through the abdomen for antibody production, purifying the generated ascites by using Protein A/G, and using the ascites for subsequent detection;
s7, antibody verification: ELISA, western blotting, co-immunoprecipitation, mass spectrometry, antibody chip and other verification are performed on the obtained monoclonal antibody cell strain to determine the most effective antibody.
As a preferred technical scheme of the invention: the Balb/c mice in the S2 are 8-12 week old Balb/c mice.
As a preferred technical scheme of the invention: and the serum titer gridlines in the S3 are serum titers of more than 10K.
As a preferred technical scheme of the invention: the fusion process in S4 is optimized PEG fusion.
As a preferred technical scheme of the invention: the number of cells per well on 4 384 well plates in S4 is 10 2 ～10 4 。
As a preferred technical scheme of the invention: the ELISA verification in S7 specifically comprises the following steps: coating an antibody to be detected on a 96-hole ELISA plate, incubating, washing, blocking the skimmed milk overnight, washing with PBS, and preserving at 4 ℃ for later use; then, antigen incubation and PBS washing are carried out, a control is arranged at the same time, his-HRP and TMB are added for color reaction, and the enzyme label instrument reads.
As a preferred technical scheme of the invention: the antibody chip in the S7 is specifically: the anti-CD 326 antibody and the control antibody are spotted on a glass sheet with NC film as a matrix by using a chip spotter to form antibody spots with the diameter of 100um, the whole protein of A375 is subjected to biotin labeling, incubated on the antibody chip according to the concentration of 2ug/ml, incubated for half an hour at room temperature, and then washed with PBS for three times, incubated with CY3-SA fluorescent secondary antibody, washed with PBS for three times, and the chip is scanned by using a GenePix fluorescent chip scanner with 523 nm.
As a preferred technical scheme of the invention: the monoclonal antibody variable region sequencing result of the mouse anti-cell surface glycoprotein CD326 is as follows:
VHF1
QIQLVQSGPELKKPGETVKISCKAS
VHCDR1
GYTFINYG
VHF2
MNWVQQAPGKGLKWMGW
VHCDR2
INTYTGEA
VHF3
TYGDDFKGRFDLSLETSASTAYLQINNLKNEDMATYFC
VHCDR3
VRFVNPMDY
VHF4
WGQGTSVTVSS
VLF1
DIVLTQSPASLAVSLGQRATISYRAS
VLCDR1
KSVSTSGYSY
VLF2
MHWNQQKPGQPPRLLIY
VLCDR2
LVS
VLF3
NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC
VLCDR3
QHIRELT
VLF4
FGGGTKLEIK。
the monoclonal antibody of the mouse anti-cell surface glycoprotein CD326, which can be applied to tumor cell capture, has the characteristics of high affinity, high specificity, multiple application scenarios and the like, and can be applied to living cell enrichment.
Drawings
FIG. 1 is a graph showing the results of antibody flow cytometry in a preferred embodiment of the present invention;
FIG. 2 is a graph showing the killing result of cancer cells according to the preferred embodiment of the present invention;
FIG. 3 is a graph showing the results of an antibody chip test in a preferred embodiment of the present invention.
Detailed Description
It should be noted that, under the condition of no conflict, the embodiments and features in the embodiments may be combined with each other, and the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1, a preferred embodiment of the present invention provides a monoclonal antibody of the mouse anti-cell surface glycoprotein CD326, which is applicable to tumor cell capturing, and the process comprises the following steps:
s1, protein QC: purchasing commercial proteins; specifically, the purchase in this embodiment is the goods number of the ACRO company: EPM-H5223-100 protein.
S2, animal immunization: selecting 6 Balb/c mice for antigen immunization, monitoring serum titers of the Balb/c mice to determine optimal immunized mice, performing boosting for 3 to 4 times after primary immunization, detecting titer by taking serum of the mice after boosting, screening out qualified mice with one impact for fusion, and continuing boosting for one to two times until the titer is highest for fusion; in particular, optimized adjuvants and immunization methods are capable of producing high affinity antibodies (IgG subtypes) against most antigens.
S3, serum detection and screening: immunized mice are subjected to orbital blood collection, and the serum titer is detected by ELISA, so that the qualified serum titer is determined;
s4, fusion and screening: fusing the whole spleen and 1/2 lymph nodes with a myeloma SP2/0 cell line, paving fused cells on 4 384-well plates for culture, collecting supernatants of all the wells, screening immune proteins by ELISA, performing microscopic examination on positive Kong Zhuaidao-well plates with cells for continuous culture, collecting supernatants of all the wells after a few days of growth, detecting reaction with soluble fragment detection antigen by ELISA, further detecting protein binding of different dilutions of positive wells, and performing affinity sequencing on 20 parent clones with highest affinity for each fusion immunogen into subclones;
s5, subcloning and screening: subcloning by limiting dilution method and ELISA screening to obtain monoclonal hybridoma cells, spreading 96-well plates on the cells, culturing to cover the bottom of about 1/6, ELISA detecting the reaction of each well supernatant on immune antigen, taking two wells with high OD value and good cell state to enter the next round of subcloning, repeating the steps until the positive rate of cell strains in the wells is 100%, obtaining monoclonal cell strains at this time, immediately expanding and culturing all positive cells after the last round of subcloning, freezing one part for later use, and preparing the other part of supernatant or ascites;
s6, preparing antibody supernatant: the finally obtained monoclonal cell strain, in this example, 8 monoclonal cell strains were finally obtained, which were injected into F1 mice via the abdomen for antibody production, and the ascites produced was purified with Protein a/G and used for subsequent detection;
s7, antibody verification: ELISA, western blotting, co-immunoprecipitation, mass spectrometry, antibody chip and the like are carried out on the obtained 8 monoclonal antibody cell strains, and the most effective antibody is determined.
In this embodiment: the Balb/c mice in the S2 are 8-12 week old Balb/c mice.
In this embodiment: and the serum titer gridlines in the S3 are serum titers of more than 10K.
In this embodiment: the fusion process in S4 is optimized PEG fusion.
In this embodiment: the number of cells per well on 4 384 well plates in S4 is 10 2 ～10 4 。
In this embodiment: the ELISA verification in S7 specifically comprises the following steps: and (3) coating the antibody to be detected on a 96-well ELISA plate, incubating, washing, blocking with skimmed milk overnight, washing with PBS, and preserving at 4 ℃ for later use. Antigen incubation (gradient dilution), PBS wash, and control were set. His-HRP (antigen with His tag) was added, TMB was developed for reaction, and the microplate reader was read.
In this example, the inventors verified the affinities of 6 cell lines by ELISA affinity of S7 as follows:
it should be added that in this example we also performed flow cytometry validation of antibodies and cancer cell killing experiments, respectively.
Specifically, as shown in fig. 1, OVCAR-3 cells are used as a detection cell line, washed by PBS, digested by EDTA, transferred into a centrifuge tube, centrifuged to discard supernatant, resuspended by PBS, goat serum is blocked for 1 hour at normal temperature, primary antibody incubation is performed at a working concentration of 50ug/ml, fluorescent secondary antibody incubation is performed at a working concentration of 1:300. the results show that the antibody can be applied to flow cytometry.
As shown in fig. 2, the cancer cell killing experiment is: 100ul of cell OVCAR-3 suspension is prepared in a 96-well plate, pre-cultured for 24 hours, 10ul of anti-CD 326 antibodies with different dilution gradients are added into a culture plate, mouse secondary antibodies coupled with small molecule drugs are added into the culture plate, the culture is carried out for 3 days, 10ul of CCK-8 solution is added into each well, the culture is carried out for 1 to 4 hours, and the absorbance at 450nm is measured by an enzyme-labeled instrument. Experimental results show that the antibody has good killing effect on cancer cells OVCAR-3.
In this embodiment: the antibody chip in the S7 is specifically: the anti-CD 326 antibody and the control antibody are spotted on a glass sheet with NC film as a matrix by using a chip spotter to form antibody spots with the diameter of 100um, the whole protein of A375 is subjected to biotin labeling, incubated on the antibody chip according to the concentration of 2ug/ml, incubated for half an hour at room temperature, and then washed with PBS for three times, incubated with CY3-SA fluorescent secondary antibody, washed with PBS for three times, and the chip is scanned by using a GenePix fluorescent chip scanner with 523 nm.
The experimental results are shown in FIG. 3, and the experimental results show that the anti-CD 326 (clone 3B 20) has obvious enrichment binding effect on the target protein, the fluorescence intensity is high, and the antigen-antibody binding reaction of the control antibody does not occur.
In this embodiment: the monoclonal antibody variable region sequencing result of the mouse anti-cell surface glycoprotein CD326 is as follows:
VHF1
QIQLVQSGPELKKPGETVKISCKAS
VHCDR1
GYTFINYG
VHF2
MNWVQQAPGKGLKWMGW
VHCDR2
INTYTGEA
VHF3
TYGDDFKGRFDLSLETSASTAYLQINNLKNEDMATYFC
VHCDR3
VRFVNPMDY
VHF4
WGQGTSVTVSS
VLF1
DIVLTQSPASLAVSLGQRATISYRAS
VLCDR1
KSVSTSGYSY
VLF2
MHWNQQKPGQPPRLLIY
VLCDR2
LVS
VLF3
NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC
VLCDR3
QHIRELT
VLF4
FGGGTKLEIK。
it will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Sequence listing
<110> general public discovery of pharmaceutical technology (Shanghai) Limited
<120> monoclonal antibody against cell surface glycoprotein CD326 of mice applicable to tumor cell capturing
<141> 2023-10-10
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> PRT
<213> Artificial Sequence
<400> 1
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser
20 25
<210> 2
<211> 8
<212> PRT
<213> Artificial Sequence
<400> 2
Gly Tyr Thr Phe Ile Asn Tyr Gly
1 5
<210> 3
<211> 17
<212> PRT
<213> Artificial Sequence
<400> 3
Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly
1 5 10 15
Trp
<210> 4
<211> 8
<212> PRT
<213> Artificial Sequence
<400> 4
Ile Asn Thr Tyr Thr Gly Glu Ala
1 5
<210> 5
<211> 38
<212> PRT
<213> Artificial Sequence
<400> 5
Thr Tyr Gly Asp Asp Phe Lys Gly Arg Phe Asp Leu Ser Leu Glu Thr
1 5 10 15
Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp
20 25 30
Met Ala Thr Tyr Phe Cys
35
<210> 6
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 6
Val Arg Phe Val Asn Pro Met Asp Tyr
1 5
<210> 7
<211> 11
<212> PRT
<213> Artificial Sequence
<400> 7
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
1 5 10
<210> 8
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 8
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser
20 25
<210> 9
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 9
Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr
1 5 10
<210> 10
<211> 17
<212> PRT
<213> Artificial Sequence
<400> 10
Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile
1 5 10 15
Tyr
<210> 11
<211> 3
<212> PRT
<213> Artificial Sequence
<400> 11
Leu Val Ser
1
<210> 12
<211> 36
<212> PRT
<213> Artificial Sequence
<400> 12
Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly
1 5 10 15
Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala
20 25 30
Thr Tyr Tyr Cys
35
<210> 13
<211> 7
<212> PRT
<213> Artificial Sequence
<400> 13
Gln His Ile Arg Glu Leu Thr
1 5
<210> 14
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 14
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
1 5 10

Claims (1)

1. A monoclonal antibody of the mouse anti-cell surface glycoprotein CD326 applicable to tumor cell capture, characterized by the variable region sequencing results of:
VHF1
QIQLVQSGPELKKPGETVKISCKAS
VHCDR1
GYTFINYG
VHF2
MNWVQQAPGKGLKWMGW
VHCDR2
INTYTGEA
VHF3
TYGDDFKGRFDLSLETSASTAYLQINNLKNEDMATYFC
VHCDR3
VRFVNPMDY
VHF4
WGQGTSVTVSS
VLF1
DIVLTQSPASLAVSLGQRATISYRAS
VLCDR1
KSVSTSGYSY
VLF2
MHWNQQKPGQPPRLLIY
VLCDR2
LVS
VLF3
NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC
VLCDR3
QHIRELT
VLF4
FGGGTKLEIK。
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Publication number Priority date Publication date Assignee Title
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JP2014105159A (en) * 2012-11-22 2014-06-09 Nippon Koden Corp Monoclonal antibody to human-derived epithelial cell adhesion molecule and method for detecting circulating tumor cells using the same
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CN101899112A (en) * 2010-03-09 2010-12-01 中国人民解放军第四军医大学 Light chain and heavy chain variable region of FMU-EPCAM-2D7 monoclonal antibody
JP2014105159A (en) * 2012-11-22 2014-06-09 Nippon Koden Corp Monoclonal antibody to human-derived epithelial cell adhesion molecule and method for detecting circulating tumor cells using the same
CN103275226A (en) * 2013-06-09 2013-09-04 中国科学技术大学 Preparation, identification and application of monoclonal antibodies of specific anti-human EpCAMs (epithelial cell adhesion molecules)
CN107383199A (en) * 2017-03-15 2017-11-24 艾比玛特医药科技（上海）有限公司 A kind of monoclonal antibody of S adenomethionine synthases and its application

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Title
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噬菌体展示EpCAM单链抗体及免疫原性初步研究;刘晓丽等;第三军医大学学报;第33卷(第10期);977-979 *
肝癌细胞单克隆抗体磁珠对人肝癌、胃癌细胞株特异性富集的实验研究;郝文斌等;现代检验医学杂志;第32卷(第06期);132-135 *

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